qPCR provides an ideal method for the rapid quantification of ocular gene expression. By carefully considering tissue-specific problems, such as light sensitivity, cellular heterogeneity as well as the consideration of suitable sample sizes based upon known or expected biological variation, researchers may save time and resources, enabling a single sample set to be used for quantification of anything up to a hundred or more transcripts.
Working along similar guidelines, qPCR may be successfully adapted for the relative quantification of gene expression in any tissue, and the underlying principles of assay design, data analysis and normalization of gene expression are universally applicable. Consideration of such fundamental aspects of qPCR assays also highlight potential sources of error, such as the effects of using different amplification efficiencies, threshold values and blind reliance on single internal control genes for normalization.
Finally, the use of kinetic analysis of qPCR data enables a high-throughput approach suitable for the rapid quantification of multiple transcripts, enabling additional measures of quality control to be incorporated. This approach is ideal for examining extended signaling pathways and validation of micro-array data, enabling researchers to move from tissue to gene expression data with unparalleled speed.
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